DE2943582C2 - L-prolyl-L-phenylalanyl-L-arginine derivatives, process for their preparation and their use - Google Patents

Description

CO -HN CO -HN COOR ₂

15th

where Ri is hydrogen or benzoyl and R _{2 is} naphthyl or 6-bromo-2-naphthyl

2. Authoring to establish the connections according spoke!, characterized in that one in each case in a manner known per se

A) an L-prolyl-L-phenylalanine derivative of the general formula II:

(Π)

30th

35

in which Rj denotes benzoyl or an amino protecting group, with an L-arginine derivative of general formula III

R ₄ -N

(CHj) ₃

NH

NH-R ₄

H ₂ N COOR ₂

45

50

wherein R _{2 has} the meanings given in claim 1 and R _{4 stands} for an amino protective group, condensed and B) from the compound of the general formula IV:

NH

in which R ₂ , Rs and R _{4 have} the meanings mentioned, splitting off the amino protective group (s)

3. Use of the L-prolyl-L-phenylalanyl-L-argine derivatives according to claim 1 as substrates for Determination of the activity of thrombin, trypsin, plasmin, kallikrein, urokinase and Cl -esterase.

The invention relates to prolylphenylalanylarginine derivatives, a process for their preparation and their Use for determining the activity of the enzymes mentioned in claim 3, the compounds serving as a substrate.

So far there are numerous methods for determining the enzyme activity. One method is that using an alkyl ester of an amino acid as a substrate brings together an enzyme and determines the enzyme activity based on the degree of hydrolysis of the alkyl ester An example of this method is the well-known Hestrin method. This is a Procedure in which an enzyme is brought into contact with an alkyl ester of an amino acid, according to after a specified period of time, the remaining ester groups are converted into hydroxamic acid with hydroxyl «min, lets this react with iron-III-chloride to develop a color and measures the color as absorbance This determines the ability of the enzyme to hydrolyze the ester and thus results from the Absorbance also includes enzyme activity

From anal. Biochem. 61,1974 200-208 (CA: 82 1975, 12 776) it is known to determine plasmin by means of "-N-methyl-Ä-N-tosyl-L-lysine - /? - naphthyIester by measuring the released jJ-naphthol. There is also a Process in which a p-nitroanilide is used as substrate and the ability to hydrolyze this Connection measures (see. DE-OS 25 27 932).

In these procedures, a considerable amount of the enzyme is required and when the enzyme concentration is low or the enzyme has low activity possesses, the measurement of the enzyme activity encounters difficulties.

Compounds suitable for determining the enzyme activity should meet the following three conditions: They have compared to the enzymes an affinity, the amount of which must be easily determinable, and the determinability must be good.

The invention is based on the object of creating new amino acid derivatives that meet these requirements fulfill.

According to the invention, L-prolyl-L-phenylalanyl-L-arginine derivatives of the general formula I are created:

R ₄ -N

(CH ₂ ),

NH-R ₄

NH

CO-HN CO-HN

65

I CO —HN CO —HN COOR ₂ 'COOR ₂ " ^J (I)

(IV) where R ^{1 is} hydrogen or benzoyl and R ²

Means naphthyl or 6-bronv2-naphthyl,

The invention also provides a process for the preparation of the L-prolyl-L-phenylalanyl-L-arginine derivatives of the general formula I, which is characterized in that in each case in a manner known per se

A) an L-ProIyl-L-phenylalanine derivative of the general Formula II:

I COOH R.

(V)

I.
R ₃

wherein R _{3 has the} same meaning as before, with a compound of the general formula Vl:

(Π)

(VI)

H ₂ N COOR ₅

I CO -HN
R ₃

wherein R _{5 is} alkyl, to an ester of the general

in which R ₃ denotes benzoyl or an amino protective group 20 _{Qen formula} ym condensed with an I ^ -arginine derivative of the general formula III:

NH

R ₄ -N

(CH ₂ ),

NH - R ₄

(DD

I I

CO-HN

(VTQ

COORs

H ₂ N COOR ₂

stands, to a compound of the general formula

wherein R _{2 has} the meaning given in claim 1 wherein R ₃ and R _{5 have the} same meanings as before and R _{4 have} an amino protective group ³⁵ , and then the ester of the general

Formula VII hydrolyzed

The starting arginine derivatives of the formula III include N ^G , N ° -dibenzyloxycarbonyl-L-arginine-1-naphthyl ester trifluoroacetate, N ^G , N ^G -dibenzyloxycarbonyl-L-arginine-e-bromo ^ -naphthyl ester trifluoroacetac, and the like. These compounds can be prepared by adding an arginine derivative III 'which has a suitable protecting group and which corresponds to the following formula:

R ₄ -N

NH

NH-R ₄

45

I CO-HN '^ CO-ΗΝ' ^ COOR ₂

NH

(IV)

(CH ₂ ), ^ΝΗ ~ ^R <(πΐ <)

condensed and

from the compound of the general formula IV, in which R ₂ , R3 and R _{4 have} the meanings given above, the arinoprotecting group (s) are split off.

55

The invention also relates to the use of the compounds according to the invention as substrates for Determination of the activity of thrombin, trypsin, plasmin, kallikrein, urokinase and Cl-esterase.

As a starting compound II for the preparation of the compounds according to the invention of the general Formula I can be any of those described in Chem. Pharm. BuIU Volume 20, pages 664 to 668 Insert connections. The starting connections can also be made by connecting the all my Formula Vee:

Ri-HN COOH

wherein R _{4 has the} same meaning as before and R _{4 represents} an amino protective group different from the group R ₄ , naphthylated to form a compound of the general formula III ":

60

65 R ₄ -N

(CH ₂ ),

NH

NH-R ₄

Ri-HN

(ΠΓ)

COOR,

where R2, R. » and R «· same meanings as before own and then only the amino protective group in the «position from compound III" removed.

To prepare the compound IV, the ί compound II and the arginine derivative III are dissolved in a suitable solvent and an activating agent which is normally used, for example dicyclohexylcarbodiimide (DCC), diphenylphosphoryl azide (DPPA), an alkyl chlorocarbonate or the like, is added to the resulting solution. If necessary, a base is then added. for example, triethylamine or the like, and stir the resulting mixture to prepare Compound IV. To the solvents used include conventional solvents y> medium, for example, chloroform, dichloromethane, dimethylformamide, tetrahydrofuran and the like, insofar as the starting materials are soluble therein. The reaction temperature may range from 0 to 40 ⁰ C.

After the reaction has ended, the compound IV can be removed from the reaction mixture in a conventional manner Way isolate. When using DCC as an activating agent, the precipitated dicyclohexylurea becomes (DCU) is removed by filtration and a suitable extraction solvent is added, for example Ethyl acetate, to the filtrate. The extract is then saturated with an aqueous citric acid solution aqueous sodium bicarbonate solution and / or a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate. It is then evaporated under reduced pressure to remove the solvent and recover compound IV.

The amino protecting group of compound IV is removed in the usual manner. If the Amino protective group is benzyloxycarbonyl, the compound IV is dissolved in a suitable one Solvent and is a catalyst, for example palladium-on-charcoal or the like, to obtained solution to remove the protective group reductively. You can also use the compound IV to a Add a solution of hydrobromic acid in acetic acid and the precipitated hydrobromide salt of the desired Collect the compound by filtering to obtain the compound I.

The compounds of the general formula I according to the invention are used as substrates for determining the Activity of thrombin, trypsin, plasmin, kallikrein, urokinase and CI-esterase used. If you have the according to the invention. Bringing compound of general formula I with the enzyme in contact, serves the Compound as a substrate and through hydrolysis with the enzyme, naphthol or 6-bromo-2-naphthol is released. After a predetermined period of time, the amount of naphthol or 6-bromo-2-naphthol is measured by the Determine enzyme activity. The fact that enzyme activity can be measured extremely easily is for the quantitative analysis of an enzyme preparation, for diagnosis by measuring the enzyme level in the Blood, the diagnosis by measuring the enzyme concentration in the blood or urine, or the like, extremely important.

When determining the enzyme activity, the enzyme will be with a given amount of the compound of the general formula i brought into contact in a suitable buffer solution, and according to a predetermined The amount of naphthol or 6-bromo-2-naphthol released is the amount of time at a given temperature measured to determine the enzyme activity. The buffer solution can be a suitable one Act buffer solution at optimal pH for the enzyme. The reaction can take place under suitable constants Conditions of temperature and time are carried out, although it is preferred at one Temperature of 25 to 37 ° C after 30 minutes the amount of released naphthol or 6-bromo-2-naphthol measure up.

The amount of naphthol or 6-bromo-2-naphthol can be measured by any known method take place, for example by physico-chemical methods, z. B. Gas Chromatography. Thin layer chromatography or the like, or by chemical Methods, for example by the ice-cream chloride reaction. the diazo coupling reaction. the Fast-Violct-B-Salt (FVB) method, or similar. In terms of simplicity and determination sensitivity is one Preferred method of adding the FVB to the reaction mixture to develop color and the absorbance is determined using a photometer.

If one measures the enzyme activity and here L-prolyl-L-phenylalanyl-L-arginine-1-naphylester as When the substrate is used, the determination sensitivity is greater than when the activity is achieved using N ^ -Betvoyl-L-arginine ethyl ester or N'-tosyl-L-arginine methyl ester (TAME) is determined, which has been used as a substrate for enzymes according to the Hesterin-Methocle became. In particular, the L-prolyl-L-phenylalanyl-L-arginine naphthyl ester a mood sensitivity to Kailikrein that is about 160 times higher than that of N ^ -Benzoyl-L-arginine ethyl ester or of N'-tosyl-L-arginine methyl ester and which about 15 times larger than that of L-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide is.

The amount of naphthol or 6-bromo-2-naphthol determined using the method mentioned corresponds the enzyme activity.

With the compounds according to the invention, a change in the enzyme concentration in the blood or Easily detect urine due to various diseases. For example, there is a Relationship between significant hypertension and kallikrein activity in the urine of a hypertensive patient Person and it is known that significant hypertension by measuring the kallikrein activity can be diagnosed. So far, the following methods have been used to measure kallikrein activity applied to urine:

1. Test with TAME (Tohoku J: Med. 116 (1975) by Masahide Seino);

2. Bioassay (Tohoku J: Exp. Med. 87 (1965) by Keishi Abe);

3. Radioimmunoassay.

These methods are complicated to perform and the results fluctuate widely. About that in addition, the methods are costly and often inconvenient for clinical examination. If against it using the compounds of the general formula I according to the invention, kallikrein activities can be im Determine urine from a small amount of urine with a simple operation.

The method for determining the enzyme activity can not only be applied to a system, which only

contains a single enzyme, but is also useful for systems with several different enzymes. That means that the measurement of the enzyme distribution in the urine or blood to diagnose diseases of Of interest, however, the conventional methods have not become very common because of their complexity carried out. According to the invention, however, these complexities and difficulties are avoided. if For example, the activity of an enzyme contained in the urine is measured, an enzyme level or determine an enzyme composition in the urine that has not yet been determined. This is done by giving urine on a suitable support, separates the enzymes from each other by electrophoresis or the like, immerses the Enzymes in an aqueous solution of the compound I for a suitable period of time and then gives the aforementioned color developing agent to it.

The invention is explained in more detail by means of the examples and the drawings. Fig. I shows standard curves the activity of kaiiikrein in the human Ui in. Figure 2 is a graph showing the Shows the effectiveness of kallikrein in human urine and FIG. 3 shows that obtained by isoelectrophoresis Enzyme scheme in the urine.

example 1

Production of L-prolyl-L-phenylalanyl-L-arginine-1 naphthyl ester dihydrochloride

3% mg of N-benzyloxycarbonyl-L-prolyl-L-phenylalanine and 682 mg of N ^G , N ^G -dibenzyloxycarbonyl-L-argiriine-1-naphthyl ester trifluoroacetate are dissolved in 5 ml of dimethylformamide (DMF) and 268 dicyclohexylcarbodiimide (DCC ), 135 mg of 1-hydroxybenzotriazole (HOBt) and 0.14 ml of triethylamine (TEA) with ice cooling and then the mixture is stirred at the same temperature for 3 hours. The temperature is allowed to rise to room temperature and the mixture is then stirred for a further 24 hours. The dicyclohexylurea crystals (DCU) precipitated after the reaction are removed by filtration and ethyl acetate is added to the filtrate. The mixture is washed with 10% aqueous citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and then evaporated under reduced pressure to remove the solvent. The white powder precipitated in this way is then collected and recrystallized from chloroform / ether, 600 mg (yield 63%) of N-benzyloxycarbonyl-L-prolyl-L-phenylalanyl-Nf'.NC'-dibenzyloxycarbonyl-L-arginine- 1-naphthyl ester with melting point 164 to 167 ° C is obtained

IRvJ ^ cm- ·:

3300,2925,1745,1710,1630.
NMRo ppm (DMSOd ₆ ):

13-22 (8H, broad), 3.0 (2H, m),

4.0-4.4 (4H, broad), 4.7 (2H, broad),

4.9 (2H, broad), 5.1 (2H χ 2, s), 53 (2H, s),

7.0-8.4 (aromatic protons).

500 mg of the above ester are dissolved in 10 ml of DMF and 200 mg of 10% palladium-on-charcoal (Pd-C) and 1.0 g of a hydrochloric acid-dioxane solution (110 mg HCl / g) are added to the resulting solution, and the mixture is then stirred the mixture obtained for 2 hours at room temperature while bubbling hydrogen gas. After the reaction has ended, the Pd-C is removed by filtration and 40 ml of anhydrous diethyl ether are added to the filtrate, whereupon an oily substance precipitates. The supernatant liquid is removed by decanting. Priority 40ml anhydrous diethyl ether to the residue and the mixture is stirred to give 200 mg (Ausbeule 61%) of L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester dihydrochloride as a white powder with melting point 87 to 90 ⁰ C. (Decomp.) Receives.

IRvJ; ¹ , ¹ : cm- ':

3350.1750.1650.

^{The N (} \ N ⁽ '-dibenzyloxycarbonyl- [..- arginine-1-naphthyl ester trifluoroacetate used as starting material is produced as follows:

9.1 g of NM-methoxybenzyloxycarbonyl-N ^{t are dissolved;} , N ⁽ '-dibenzyloxycarbonyl-L-arginine (Z. Naturf. 20b. 429 (1965), F. Weygand, E. Nintz) in 30 ml of anhydrous FytiuiM iiiiu gifoi 7.3 g!, I'-Dinuph'hy' Sülfit 7MT LcS'Jüg.

The mixture obtained is stirred for 24 hours at room temperature. To the reaction mixture was added ethyl acetate and the mixture is washed with a 10% citric acid solution, a saturated sodium r-, umbicarbonatlösung and a saturated Natriumcnloridlösung in this order, dried over anhydrous magnesium sulfate and then evaporated under reduced pressure to dryness. Anhydrous is added to the residue: diethyl ether is added to wash it, giving 9.0 g (yield 82%) of a pale yellow, viscous, oily material, which is the NM-methox> benzyloxycarbonyl-N ^G , N ^Ü -dibenzyloxycarbonyi-L-arginine-1-naphihylcster acts.

I Rr Jj ?; cm - ':

3400,1760,1720,1690.
NMRo ppm (CDCI ₃ ):

1.9 (4H, m), 3.7 (3H.s), 4, l (2H, m),

4.8 (1H, m), 5.1 (2H χ 2, s), 5.2 (2H, s),

6.7-8.0 (aromatic protons).

15 ml of trifluoroacetic acid are added to 8.0 g of the above ester, and the mixture is stirred for 30 minutes under ice cooling. After the reaction has ended, the trifluoroacetic acid is removed by evaporation reduced pressure and anhydrous diethyl ether is added to the residue in order to dissolve it. Thereafter the solution obtained is allowed to stand. The colorless, needle-shaped crystals precipitated in this way are collected. 4.5 g are obtained (yield 61%)

N ^G , N ^G -dibenzyloxycarbonyl-L-arginine-1-naphthyl ester trifluoroacetate with melting point 149 to 151 ° C.

I Rv J;?, 'Cm -':
3400,1760,1720,1660.

NMR 6 ppm (CDCl ₃ + DMSOd ₆ ):

2.1 (4H, m), 4.0 (2H, m), 4.5 (I H, m), 5.1 (2H, s), 5.2 (2H, s), 7.1-8.0 (aromatic protons).

Example 2

Preparation of Benzoyl-L-prolyl-L-phenylalanyl-L-arginine-1 -naphthyiester hydrochloride

752 mg of benzoyl-L-prolyl-L-phenylalanine are dissolved and 1364 g of N ^ N ^ -dibenzyloxycarbonyl-L-arginine-inaphthyl ester trifluoroacetate in 10 ml DMF and then add 535 mg DCC, 270 mg HOBt and 0.28 while cooling with ice

ml TEA to the resulting solution. Then stir for 3 hours. The temperature is then allowed to rise to room temperature and the mixture is stirred for a further 24 hours at room temperature. When the reaction has ended, the DCU which has precipitated out in this way is removed by filtration and ethyl acetate is added to the filtrate. The obtained mixture is washed with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in that order, dried over anhydrous magnesium sulfate, and then evaporated under reduced pressure to remove the solvent. The residue is recrystallized from ethyl acetate / diethyl ether / n-hexane, 1.20 g (yield 62%) of a white powder having a melting point of 140 ° to 143 ° C. which is benzoyl-L-proljl-L -phenylalanyl-N ^G , N ^G -dibenzyloxycarbonyl-L-arginine-1-naphthyl ester.

20th

IR * ™ 'crn-':

3400,3300.1750,1720,1640,1610.

NMR ό ppm (CDCl ₃ ):

1.6-2.2 (8H. Broad), 3.2 (2H, m), 3.5 (2H, m), 4.0 (2H, m), 4.4-5.0 (3H, m). 5.2 (2H, s). 5.3 (2H, s), 7.0-8.0 (aromatic protons).

962 mg of the above ester are dissolved in 10 ml of DMF and then 500 mg of 10% Pd-C and 0.5 ml of water-chloride are added material acid-dioxane solution (98 mg HCl / g) to the obtained Solution. The mixture is stirred for 3 hours at room temperature while bubbling hydrogen gas. When the reaction has ended, the Pd-C is filtered off and 100 ml of anhydrous diethyl ether are added to the filtrate, J5 whereupon an oily substance precipitates. The supernatant liquid is removed by decantation and one add anhydrous diethyl ether to the residue and stir the mixture. This gives 600 mg (Yield 87%) of a white powder with a melting point -to 86 to 90 ° C (decomp.), Which is benzoyl-L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester hydrochloride acts.

45

3600,1750,1650.

The benzoyl-L-prolyl-L-phenylalanine used as starting material is made in the following way:

10.7 g of benzoyl-L-proline and 11.5 g are dissolved L-phenylalanine ethyl ester hydrochloride in 100 ml dichloromethane and add 153 g DPPA and 15 ml TEA with ice cooling. Then the mixture is 24 Stirred for hours. The reaction mixture is washed with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in that order and then over-drying anhydrous magnesium sulfate. It is then evaporated under reduced pressure to remove the solvent to remove. The residue is recrystallized from ethyl acetate, 1.20 g (yield 62%) of a white powder with a melting point of 133 to 135 ° C, which is the benzoyl-L-prolyl-L-phenylalanine ethyl ester acts.

12.0 g of the above ester are dissolved in 100 ml of methanol and 45 ml of an IN sodium hydroxide solution are added, whereupon the mixture is stirred for 5 hours. The methanol is removed at low temperature and ethyl acetate and distilled water are added to the residue and then the resulting mixture is shaken. The aqueous phase is made weakly acidic with 10% hydrochloric acid solution and the crystalline powder precipitated in this way is collected. 10.6 g (yield 95%) of benzoyl-L-prolyl-L-phenylalanine with a melting point of 200 to 202 ^° C. are obtained.

3375,1730,1670.

Example 3

Preparation of Benzoyl-L-prolyl-L-phenylalanyl-L-arginine-6-bromo-2-naphthyl ester hydrochloride

376 mg of benzoyl-L-prolyl-L-phenylalanm and 761 mg of N ^G .N ^(; -dibenzyloxycarbonyl-L-arginine-uhrom-2-naphthyl ester trifluoroacetate are dissolved in 10 ml of DMF and then 267 mg of DCC, 135 mg of HOBt and 0.17 ml TEA. The mixture is then stirred for 3 hours and then for a further 24 hours at room temperature. The DCU precipitated in this way is removed by filtration, and ethyl acetate is added to the filtrate. The mixture is then saturated with 10% citric acid solution Sodium bicarbonate solution and saturated sodium chloride solution are washed in this order and then dried over anhydrous magnesium sulfate. The residue is removed from ethyl acetate / diethyl ether. In this way, 500 mg (yield 50%) of a white powder with a melting point of 94 to 97 ° C. are obtained is the benzoyl-L-prolyl-L-phenylalanyl-N ^G , N ^G -dibenzyloxycarbonyl-L-arginine-6-bromo-2-naphthyl ester.

IRvL *; cm- ¹ :

3375,1750,1710,1610.

0.5 g of the above ester is dissolved in 5 ml of DMF and then 250 mg of 10% Pd-C and 250 mg of a hydrochloric acid / dioxane solution are added (98 mg HCl / g) zii. One stirs the mixture for 3 hours at room temperature while passing hydrogen gas through. After the reaction has ended the reaction mixture is filtered in order to remove the Pd-C and 100 ml of anhydrous diethyl ether are added to the filtrate. The white powder precipitated in this way is collected. 210 mg (yield 38%) Benzoyl-L-prolyl-L-phenylalanyl-L-arginine-öbromo-2-naphthyl ester hydrochloride with a melting point of 80 to 85 ° C.

IRvS ?; cm-':

3350,1750,1650.

Example 4

Preparation of L-prolyl-L-phenylalanyl-L-arginine-6-bromo-2-naphthyl ester dihydrochloride

396 mg of N-benzyloxycarbonyl-L-prolyl-L-phenylalanine and 761 mg of N ^G , N ^G -dibenzyloxycarbonyl-L-arginine-6-bromo-2-naphthyl ester trifluoroacetate are dissolved in 10 ml of DMF and then added while cooling with ice 267 mg DCC, 135 mg HOBt and 0.17 ml TEA. The mixture is stirred with ice-cooling for 3 hours and for a further 25 hours at room temperature. The DCU which has precipitated out in this way is removed by filtration, and

ethyl ester is added to the filtrate. The obtained mixture is washed with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in that order, dried over anhydrous magnesium sulfate, and then evaporated under reduced pressure to remove the solvent. Diethyl ether is added to the residue and the mixture obtained is stirred, 610 mg (yield 59.5%) of a white powder having a melting point of 125 to 130 ^° C., which is N-benzyloxycarbonyl-L-prolyl-L- phenylalanyl-NC'.NG-dibenzyloxycarbonyl-L-arganine-ebrorti-i-naphthyl ester.

IR ^v m? <Cm- ':

3400, 3300, 1750, 1710, 1650, 1610.

NMRO ppm (CDCl ₃ ):

1.5-2.2 (8H, broad). 3.1 (2H, m), 3.4 (2H. M), 4.0 (3H, in). 4.7 (2H, m), 5.1 (2H, s), 5.2 (2H, s), 5.3 (2H, s),

Dissolve 510 mg of the above ester in 5 ml of DMF and then gives 250 mg of 10% Pd-C and 440 mg of hydrochloric acid Dioxane solution (98 mg HCl / g). The mixture is then stirred for 3 hours at room temperature, while bubbling hydrogen gas. After the reaction has ended, the Pd-C is removed by filtration and add 100 ml of anhydrous diethyl ether to the filtrate. The mixture is left under ice-cooling for 24 hours stand. The supernatant liquid is removed by decantation and diethyl ether is added again to the residue. The mixture is then stirred, 230 mg (yield 67%) of a white powder with Melting point 80 to 84 ° C, which is L-prolyl-L-phenylalanyl-L-arginine-6-bromo-2-naphthyl ester dihydrochloride acts.

cm-':
3350,1750,1655.

Example 5

Measurement of the activity of kallikrein under

Use of L-prolyl-L-phenylalanyl-L-arginine-1 -naphthyl ester dihydrochloride as substrate

Add to 1.7 ml of 50 mM phosphate buffer solution (pH 7.0) one 0.1 with an aqueous solution of kallikrein from human urine in various concentrations (U; 2.4; 3.6 and 4.8 χ 10- * esterase units (EU) Kallikrein) and 0.2 ml of a 1.5 mM solution of L-prolylphenyl-L-alanyl-L-arginine-1-naphthyl ester dihydrochloride, dissolved in 0.15% sodium lauryl sulfate solution. Thereafter, the mixture is kept at 37 ° C for 30 minutes incubated 20 microliters of an 8% sodium lauryl sulfate solution are added to the mixture and the resulting solution is cooled Mix with ice water. 0.2 ml of a 1% strength FVB solution are added to the mixture and the Stand mixture for 10 minutes at 0 ° C. Then 2ml glacial acetic acid is added. Developed in this way Color is measured as absorbance (505 nm) using a Spectrophotometer measured to determine the amount of released naphthol. As a control the same, kallikrein-free buffer solution is used. The amount of released naphthol corresponds to Enzyme activity

The results obtained at each concentration of kallikrein using the above procedure are in Fig. 1 shown

When using Benzoyl-L-prolyl-L-phenylala-

nyl-L-arginine-l-naphthyl ester hydrochloride was the Absorbance measured at 505 nm when using the 6-bromo-2-naphthyl ester, the extinction was on the other hand measured at 530 nm.

Comparative example 1

Measurement of the activity of kallikrein using N "-Tosyl-L-arginine methyl ester hydrochloride as a substrate

To 0.5 ml of an aqueous solution of kallikrein from the urine of which 12, 24,36 or 48 χ -F.sterase 10- ⁴ units of kallikrein contains, are to 0.4 ml π i * - Tosylargininmethylesterlösung (10 micromoles / 0.4 ml 5% DMSO) and 0.1 ml phosphate buffer (pH 7.4). The mixture is incubated for 30 minutes at 37 ^° C., and 1.5 ml of a hydroxyiamine solution (mixture of equal amounts of 2 M NH 2 O H hydrochloride and 3.5 M ! "NaGrI) / U üi" iäui däfiii die ivliSCnüPig i5 minUiCu uCi at room temperature. 1 ml of 18% trichloroacetic acid solution, 1 ml of 4N hydrochloric acid and 1 ml of 10% iron (III) chloride solution are added and the resulting mixture is stirred thoroughly and then centrifuged for 10 minutes at 3000 rpm.

The color developed in the supernatant liquid is recorded as the extinction (530 nm) using a Measured by spectrophotometer. The value obtained corresponds to the amount of substrate, which by the Kallikrein was not hydrolyzed, and thus the enzyme activity is the same as the difference between the value obtained when no enzyme was used (control) and the value obtained after the enzyme reaction was obtained.

The results obtained at each concentration of kallikrein in Example 5 and Comparative Example 1 are shown in FIG Fig. 1 shown. From Fig. 1 it can be seen that the method according to Example 5 has a sensitivity which is about 160 times the size of the method

• fo according to comparative example 1 St. In Fig. 1, curve A represents the standard curve which was obtained by the method of Example 5 using L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester dihydrochloride as substrate. Curve B means the St? Standard curve obtained by the method of Comparative Example 1. Curve C relates to benzoyl-L-Pro-L-Phe-L-Arg-1-naphthyl ester, curve D to L-Pro-L-Phe-L-Arg-6-bromo-2-naphthyl ester, and curve E to Benzoyl-L-Pro-L-Phe-L-Arg-o-bromo ^ -naphthyl ester.

For curves B, C, D and E, the numbers in brackets are units of the abscissa.

Example 6 Measurement of kallikrein in human urine

1.7 ml of 50 mM are added to 0.1 ml of human urine

Phosphate buffer solution (pH 7.0) and 0.2 ml of a 1.5 mM L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester dihydrochloride solution. The mixture is then incubated for 30 minutes at 37 ° C. The mixture is cooled to 0 ^° C. and 0.2 ml of a 1% strength FVB solution is added and the resulting mixture is then left to ^{stand for 10 minutes at 0 °} C. Subsequently, it is added 2 ml of glacial acetic acid are added and the color developed in this way is measured as the extinction (505 nm) with the aid of a spectrophotometer, which determines the amount of naphthol released by the hydrolysis with the enzyme

t3

the same procedure was repeated as before, except that the human urine after heat treatment was used to measure the absorbance. The difference in absorbance (AA) between the two measurements was obtained. This difference corresponds to the activity of kallikrein in human urine. The results of the measurement using the above method are shown in Fig. 2. Here, the urine samples were obtained from eight different male individuals (sample Nos. 1 to 8).

Example 7

Measurement of the enzyme distribution in the urine Isoelectrophoresis

A column with 4% acrylamide gel with a final concentration of 2% amphonene (pH 3.5 to 5) is prepared, by using a glass tube with an inner diameter of 9 mm and a length of 10 cm, 2 ^ ml of urine can be used Freeze-dried and dissolved in 50 microliters of water to give a sample. This sample is applied to the Above column abandoned and 4 hours at 200 V electrophoresed Then the gel is removed, with Washed water, immersed in 0.5 M phosphate buffer solution for 5 minutes, then in 0.25 M phosphate buffer solution (pH 7.0) containing 0.2 microniol L-prolyl-L-phenylalanyl-L-argamn-l-naphthyl ester and 1% FVB immersed and then incubated for 30 minutes at 37 ° C. Using the above method, observe the enzyme distribution in the urine as a pink or purple band. F i g. 3 shows the enzyne distribution at

ίο using normal human urine.

Comparative example 2

The reaction rates of the well-known oc-N-methyl-a-N-tosyl-L-lysine-ß-naphthyl esters (CA: 82, 1975, 12776p), which can be used as compound 1 is referred to, and the invention

L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester (Compound 2) for the determination of four different enzymes by the method of Example 5 compared.

As the table below shows, those with the Compound 2 according to the invention obtained compared with those of the Compound 1 much higher.

Tabel

Thrombin

Kallikrein

Plasmin

Urokinase

Connection (1) Connection (2)

2.9X10-2

1.1X10- * 0.32

Units: Thrombin: // Mol / min / IU at 2S ^a C Kallikrein: // Mol / min / KU at 2S ° C Plasmin: // mol / min / CU at 25 ° C Urokinase: ^ mol / min / lU at 25 ° C

0.14

1,2X10- « 9.6X10-6

With the compounds according to the invention, significantly higher reaction rates are achieved Hydrolysis obtained by the enzyme, which also results in -to very small enzyme activities can be determined more reliably and easily.

For this purpose 2 sheets of drawings

Claims (1)

Patent claims: 1, L-prolyl-L-phenylalanyl-L-arginine derivatives of general formula I: NH NH- / CH ₂ (CHj) ₃ ^NH,